Imaging system, methodology, and applications employing reciprocal space optical design

ABSTRACT

An imaging system, methodology, and various applications are provided to facilitate optical imaging performance. The system includes a sensor having one or more receptors and an image transfer medium to scale the sensor and receptors in accordance with resolvable characteristics of the medium. A computer, memory, and/or display associated with the sensor provides storage and/or display of information relating to output from the receptors to produce and/or process an image, wherein a plurality of illumination sources can also be utilized in conjunction with the image transfer medium. The image transfer medium can be configured as a k-space filter that correlates a pitch associated with the receptors to a diffraction-limited spot associated with the image transfer medium, wherein the pitch can be unit-mapped to about the size of the diffraction-limited spot.

RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/758,739 which was filed Jan. 16, 2004, entitled IMAGINGSYSTEM, METHODOLOGY, AND APPLICATIONS EMPLOYING RECIPROCAL SPACE OPTICALDESIGN. This application is also a continuation-in-part of U.S. patentapplication Ser. No. 10/189,326 which was filed Jul. 2, 2002 entitledIMAGING SYSTEM AND METHODOLOGY EMPLOYING RECIPROCAL SPACE OPTICALDESIGN, which is a continuation-in-part of U.S. patent application Ser.No. 09/900,218, which was filed Jul. 6, 2001, entitled IMAGING SYSTEMAND METHODOLOGY EMPLOYING RECIPROCAL SPACE OPTICAL DESIGN, both of whichare incorporated herein by reference.

TECHNICAL FIELD

The present invention relates generally to image and optical systems,and more particularly to a system and method to facilitate imagingperformance via an image transfer medium that projects characteristicsof a sensor to an object field of view.

BACKGROUND OF THE INVENTION

Microscopes facilitate creating a large image of a tiny object. Greatermagnification can be achieved if the light from an object is made topass through two lenses compared to a simple microscope with one lens. Acompound microscope has two or more converging lenses, placed in linewith one another, so that both lenses refract the light in turn. Theresult is to produce an image that is magnified with improved quality inResolved Magnification and other image parameters than either lens couldalone. Light illuminating the object first passes through a short focallength lens or lens group, called the objective, and then travels onsome distance before being passed through a longer focal length lens orlens group, called the eyepiece. A lens group is often simply referredto singularly as a lens. Usually these two lenses are held in paraxialrelationship to one another, so that the axis of one lens is arranged tobe in the same orientation as the axis of the second lens. It is thenature of the lenses, their properties, their relationship, and therelationship of the objective lens to the object that determines how ahighly magnified image is produced in the eye of the observer.

The first lens or objective is usually a small lens with a very smallfocal length. A specimen or object is placed in the path of a lightsource with sufficient intensity to illuminate as desired. The objectivelens is then lowered until the specimen is very close to, but not quiteat the focal point of the lens. Light leaving the specimen and passingthrough the objective lens produces a real, inverted and magnified imagebehind the lens, in the microscope at a point generally referred to asthe intermediate image plane. The second lens or eyepiece has a longerfocal length and is placed in the microscope so that the image producedby the objective lens falls closer to the eyepiece than one focal length(that is, inside the focal point of the lens). The image from theobjective lens now becomes the object for the eyepiece lens. As thisobject is inside one focal length, the second lens refracts the light insuch a way as to produce a second image that is virtual, inverted andmagnified. This is the final image seen by the eye of the observer.

Alternatively, common infinity space or infinity corrected designmicroscopes employ objective lenses with infinite conjugate propertiessuch that the light leaving the objective is not focused, but is a fluxof parallel rays which do not converge until after passing through atube lens where the projected image is then located at the focal pointof the eyepiece for magnification and observation. Many microscopes,such as the compound microscope described above, are designed to provideimages of certain quality to the human eye through an eyepiece.Connecting a Machine Vision Sensor, such as a Charge Coupled Device(CCD) sensor, to the microscope so that an image may be viewed on amonitor presents difficulties. This is because the image qualityprovided by the sensor and viewed by a human eye decreases as comparedto an image viewed by a human eye directly through an eyepiece. As aresult, conventional optical systems for magnifying, observing,examining, and analyzing small items often require the careful attentionof a technician monitoring the process through an eyepiece. It is forthis reason, as well as others, that Machine-Vision or computer-basedimage displays from the aforementioned image sensor displayed on amonitor or other output display device are not of quality perceived bythe human observer through the eyepiece.

SUMMARY OF THE INVENTION

The following presents a simplified summary of the invention in order toprovide a basic understanding of some aspects of the invention. Thissummary is not an extensive overview of the invention. It is intended toneither identify key or critical elements of the invention nor delineatethe scope of the invention. Its sole purpose is to present some conceptsof the invention in a simplified form as a prelude to the more detaileddescription that is presented later.

The present invention relates to a system and methodology thatfacilitates imaging performance of optical imaging systems. In regard toseveral optical and/or imaging system parameters, many orders ofperformance enhancement can be realized over conventional systems (e.g.,greater effective resolved magnification, larger working distances,increased absolute spatial resolution, increased spatial field of view,increased depth of field, Modulation Transfer Function of about 1, oilimmersion objectives and eye pieces not required). This is achieved byadapting an image transfer medium (e.g., one or more lenses, fiberoptical media, or other media) to a sensor having one or more receptors(e.g., pixels) such that the receptors of the sensor are effectivelyscaled (e.g., “mapped”, “sized”, “projected”, “matched”, “reduced”) tooccupy an object field of view at about the scale or size associatedwith a diffraction limited point or spot within the object field ofview. Thus, a band-pass filtering of spatial frequencies in what isknown as Fourier space or “k-space” is achieved such that the projectedsize (projection in a direction from the sensor toward object space) ofthe receptor is filled in k-space.

In other words, the image transfer medium is adapted, configured and/orselected such that a transform into k-space is achieved, wherein an apriori design determination causes k-space or band-pass frequencies ofinterest to be substantially preserved throughout and frequencies aboveand below the k-space frequencies to be mitigated. It is noted that thefrequencies above and below the k-space frequencies tend to causeblurring and contrast reduction and are generally associated withconventional optical system designs which define intrinsic constraintson a Modulation Transfer Function and “optical noise”. This furtherillustrates that the systems and methods of the present invention are incontravention or opposition to conventional geometric paraxial raydesigns. Consequently, many known optical design limitations associatedwith conventional systems are mitigated by the present invention.

According to one aspect of the present invention, a “k-space” design,system and methodology is provided which defines a “unit-mapping” of theModulation Transfer Function (MTF) of an object plane to image planerelationship. The k-space design projects image plane pixels orreceptors forward to the object plane to promote an optimum theoreticalrelationship. This is defined by a substantially one-to-onecorrespondence between image sensor receptors and projected object planeunits (e.g., units defined by smallest resolvable points or spots in anoptical or image transfer medium) that are matched according to thereceptor size. The k-Space design defines that “unit-mapping” or“unit-matching” acts as an effective “Intrinsic Spatial Filter” whichimplies that spectral components of both an object and an image ink-space (also referred to as “reciprocal-space”) are substantiallymatched or quantized. Advantages provided by the k-space design resultin a system and methodology capable of much higher effective resolvedmagnification with concomitantly related and much increased Field OfView, Depth Of Field, Absolute Spatial Resolution, and Working Distancesutilizing dry objective lens imaging, for example, and without employingconventional oil immersion techniques having inherent intrinsiclimitations to the aforementioned parameters.

One aspect of the present invention relates to an optical system thatincludes an optical sensor having an array of light receptors having apixel pitch. A lens optically associated with the optical sensor isconfigured with optical parameters functionally related to the pitch anda desired resolution of the optical system. As a result, the lens isoperative to substantially map a portion of an object having the desiredresolution along the optical path to an associated one of the lightreceptors.

Another aspect of the present invention relates to a method of designingan optical system. The method includes selecting a sensor with aplurality of light receptors having a pixel pitch. A desired minimumspot size resolution is selected for the system and a lens configured oran extant lens selected with optical parameters based on the pixel pitchand the desired minimum spot size is provided so as to map the pluralityof light receptors to part of the image according to the desiredresolution.

The present invention can be employed in various portable, stand-alone,or a combination of portable and stand-alone applications. For example,this can include portable imaging systems that can be distributedthroughout the world to support various remote imaging applications.Such applications can include remote medicine or industrial applicationswhereby an image is generated in one location and transmitted to anotherlocation for analysis (e.g., remote pathology application).

The following description and the annexed drawings set forth in detailcertain illustrative aspects of the invention. These aspects areindicative, however, of but a few of the various ways in which theprinciples of the invention may be employed and the present invention isintended to include all such aspects and their equivalents. Otheradvantages and novel features of the invention will become apparent fromthe following detailed description of the invention when considered inconjunction with the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic block diagram illustrating an imaging system inaccordance with an aspect of the present invention.

FIG. 2 is a diagram illustrating a k-space system design in accordancewith an aspect of the present invention.

FIG. 3 is a diagram of an exemplary system illustrating sensor receptormatching in accordance with an aspect of the present invention.

FIG. 4 is a graph illustrating sensor matching considerations inaccordance with an aspect of the present invention.

FIG. 5 is a graph illustrating a Modulation Transfer Function inaccordance with an aspect of the present invention.

FIG. 6 is a graph illustrating a figure of merit relating to a SpatialField Number in accordance with an aspect of the present invention.

FIG. 7 is a flow diagram illustrating an imaging methodology inaccordance with an aspect of the present invention.

FIG. 8 is a flow diagram illustrating a methodology for selectingoptical parameters in accordance with an aspect of the presentinvention.

FIGS. 9-17 illustrate various exemplary imaging designs in accordancewith an aspect of the present invention.

FIG. 18 illustrates an excitation application in accordance with anaspect of the present invention.

FIGS. 19 and 20 illustrate beam-scanning technologies that can beapplied to increase excitation yields in accordance with an aspect ofthe present invention.

FIG. 21 illustrates an example system for integrating or retrofittingpixel-mapping components in accordance with an aspect of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an optical and/or imaging system andmethodology. According to one aspect of the present invention, a k-spacefilter is provided that can be configured from an image transfer mediumsuch as optical media that correlates image sensor receptors to anoptical or image transfer medium. A variety of illumination sources canalso be employed to achieve one or more operational goals and forversatility of application. The k-space design of the imaging system ofthe present invention promotes capture and analysis (e.g., automatedand/or manual) of images having a high Field Of View (FOV) atsubstantially high Effective Resolved Magnification as compared toconventional systems. This can include employing a small NumericalAperture (NA) associated with lower magnification objective lenses toachieve very high Effective Resolved Magnification. As a consequence,images having a substantially large Depth Of Field (DOF) at very highEffective Resolved Magnification are also realized. The k-space designalso facilitates employment of homogeneous illumination sources that aresubstantially insensitive to changes in position, thereby improvingmethods of examination and analysis.

According to another aspect of the present invention, an objective lensto object distance (e.g., Working Distance) can be maintained inoperation at low and high power effective resolved magnificationimaging, wherein typical spacing can be achieved at about 0.1 mm or moreand about 20 mm or less, as opposed to conventional microscopic systemswhich can require significantly smaller (as small as 0.01 mm) object toobjective lens distances for comparable (e.g., similar order ofmagnitude) Effective Resolved Magnification values. In another aspect,the Working Distance is about 0.5 mm or more and about 10 mm or less. Itis to be appreciated that the present invention is not limited tooperating at the above working distances. In many instances the aboveworking distances are employed, however, in some instances, smaller orlarger distances are employed. It is further noted that oil immersion orother Index of Refraction matching media or fluids for objective lensesare generally not required (e.g., substantially no improvement to begained) at one or more effective image magnification levels of thepresent invention yet, still exceeding effective resolved magnificationlevels achievable in conventional microscopic optical design variationsincluding systems employing “infinity-corrected” objective lenses.

The k-space design of the present invention defines that a small “BlurCircle” or diffraction limited point/spot at the object plane isdetermined by parameters of the design to match image sensor receptorsor pixels with a substantially one-to-one correspondence by“unit-mapping” of object and image spaces for associated object andimage fields. This enables the improved performance and capabilities ofthe present invention. One possible theory of the k-space design resultsfrom the mathematical concept that since the Fourier Transform of bothan object and an image is formed in k-space (also called “reciprocalspace”), the sensor should be mapped to the object plane in k-space viaoptical design techniques and component placement in accordance with thepresent invention. It is to be appreciated that a plurality of othertransforms or models can be utilized to configure and/or select one ormore components in accordance with the present invention. For example,wavelet transforms, Laplace (s-transforms), z-transforms as well asother transforms can be similarly employed.

The k-space design methodology is unlike conventional optical systemsdesigned according to geometric, paraxial ray-trace and optimizationtheory, since the k-space optimization facilitates that the spectralcomponents of the object (e.g., tissue sample, particle, semiconductor)and the image are the same in k-space, and thus quantized. Therefore,there are substantially no inherent limitations imposed on a ModulationTransfer Function (MTF) describing contrast versus resolution andabsolute spatial resolution in the present invention. Quantization, forexample, in k-space yields a substantially unitary Modulation TransferFunction not realized by conventional systems. It is noted that highMTF, Spatial Resolution, and effective resolved image magnification canbe achieved with much lower magnification objective lenses withdesirable lower Numerical Apertures (e.g., generally less than about 50×with a numerical aperture of generally less than about 0.7) through“unit-mapping” of projected pixels in an “Intrinsic Spatial Filter”provided by the k-space design.

If desired, “infinity-corrected” objectives can be employed withassociated optical component and illumination, as well as spectrumvarying components, polarization varying components, and/or contrast orphase varying components. These components can be included in an opticalpath-length between an objective and the image lens within an “infinityspace”. Optical system accessories and variations can thus be positionedas interchangeable modules in this geometry. The k-space design, incontrast to conventional microscopic imagers that utilize“infinity-corrected” objectives, enables the maximum optimization of theinfinity space geometry by the “unit-mapping” concept. This implies thatthere is generally no specific limit to the number of additionalcomponents that can be inserted in the “infinity space” geometry as inconventional microscopic systems that typically specify no more than 2additional components without optical correction.

The present invention also enables a “base-module” design that can beconfigured and reconfigured in operation for a plurality of differentapplications if necessary to employ transmissive and/or reflectedillumination, if desired. This includes substantially all typicalmachine vision illumination schemes (e.g., darkfield, brightfield,phase-contrast), and other microscopic transmissive techniques (Kohler,Abbe), in substantially any offset and can include Epi-illumination—andvariants thereof. The systems of the present invention can be employedin a plurality of opto-mechanical designs that are robust since thek-space design is substantially not sensitive to environmental andmechanical vibration and thus generally does not require heavystructural mechanical design and isolation from vibration associatedwith conventional microscopic imaging instruments. Other features caninclude digital image processing, if desired, along with storage (e.g.,local database, image data transmissions to remote computers forstorage/analysis) and display of the images produced in accordance withthe present invention (e.g., computer display, printer, film, and otheroutput media). Remote signal processing of image data can be provided,along with communication and display of the image data via associateddata packets that are communicated over a network or other medium, forexample.

Moreover, images that are created in accordance with the presentinvention can be stored and/or transmitted with other digitalinformation (e.g., audio data, other images, medical histories, productinformation, analysis information, and so forth). For example, an imagemay have associated voice-encoded data describing one or more aspects ofthe image or images contained as part of a data package that can bestored locally and/or transmitted across a network for remote storageand/or further analysis. In one specific example, an image created inaccordance with the present invention can be transmitted to a remotelocation, wherein the image is further analyzed (e.g., medical orproduct specialist analyzes received image on a computer or imagedisplay). After analysis, a voice encoding or related data is appendedor encoded with the received image and then transmitted back to theoriginating location (or other location), wherein the image andresultant encoded analysis can be reviewed. As can be appreciated,substantially any type of digital information can be stored and/ortransmitted with images that are created in accordance with the presentinvention.

Also, as will be apparent from the following description, the presentinvention can be economically implemented in a plurality of variouspackages including integrated imaging/computing systems that areemployed to analyze various samples. Such systems include handhelddevices, notebook computers, laptops, personal digital assistants, andso forth that are adapted with the imaging concepts described herein.

Referring initially to FIG. 1, an imaging system 10 is illustrated inaccordance with an aspect of the present invention. The imaging system10 includes a sensor 20 having one or more receptors such as pixels ordiscrete light detectors (See e.g., illustrated below in FIG. 3)operably associated with an image transfer medium 30. The image transfermedium 30 is adapted or configured to scale the proportions of thesensor 20 at an image plane established by the position of the sensor 20to an object field of view illustrated at reference numeral 34. A planarreference 36 of X and Y coordinates is provided to illustrate thescaling or reduction of the apparent or virtual size of the sensor 20 tothe object field of view 34. Direction arrows 38 and 40 illustrate thedirection of reduction of the apparent size of the sensor 20 toward theobject field of view 34.

The object field of view 34 established by the image transfer medium 30is related to the position of an object plane 42 that includes one ormore items under microscopic examination (not shown). It is noted thatthe sensor 20 can be substantially any size, shape and/or technology(e.g., digital sensor, analog sensor, Charge Coupled Device (CCD)sensor, CMOS sensor, Charge Injection Device (CID) sensor, an arraysensor, a linear scan sensor) including one or more receptors of varioussizes and shapes, the one or more receptors being similarly sized orproportioned on a respective sensor to be responsive to light (e.g.,visible, non-visible, “light”, “radiation”, or other such “visible” or“invisible” or “non-visible” hereafter meaning radiation of some desiredwavelength optically directed. That is: radiation of any particularwavelength whose optical path, direction, and/or path length is alteredby means of an optical medium, surface, material, component, orcomponents, or other such means suitable to radiation of that wavelengthin the configuration or configurations pertaining to the direction ofsuch radiation to achieve the desired characteristics in accordance withthe present invention) received from the items under examination in theobject field of view 34.

As light is received from the object field of view 34, the sensor 20provides an output 44 that can be directed to a local or remote storagesuch as a memory (not shown) and displayed from the memory via acomputer and associated display, for example, without substantially anyintervening digital processing (e.g., straight bit map from sensormemory to display), if desired. It is noted that local or remote signalprocessing of the image data received from the sensor 20 can also occur.For example, the output 44 can be converted to electronic data packetsand transmitted to a remote system over a network and/or via wirelesstransmissions systems and protocols for further analysis and/or display.Similarly, the output 44 can be stored in a local computer memory beforebeing transmitted to a subsequent computing system for further analysisand/or display.

The scaling provided by the image transfer medium 30 is determined by anovel k-space configuration or design within the medium that promotespredetermined k-space frequencies of interest and mitigates frequenciesoutside the predetermined frequencies. This has the effect of aband-pass filter of the spatial frequencies within the image transfermedium 30 and notably defines the imaging system 10 in terms ofresolution rather than magnification. As will be described in moredetail below, the resolution of the imaging system 10 determined by thek-space design promotes a plurality of features in a displayed or storedimage such as having high effective resolved magnification, highabsolute spatial resolution, large depth of field, larger workingdistances, and a unitary Modulation Transfer Function as well as otherfeatures.

In order to determine the k-space frequencies, a “pitch” or spacing isdetermined between adjacent receptors on the sensor 20, the pitchrelated to the center-to-center distance of adjacent receptors and aboutthe size or diameter of a single receptor. The pitch of the sensor 20defines the Nyquist “cut-off” frequency band of the sensor. It is thisfrequency band that is promoted by the k-space design, whereas otherfrequencies are mitigated. In order to illustrate how scaling isdetermined in the imaging system 10, a small or diffraction limited spotor point 50 is illustrated at the object plane 42. The diffractionlimited point 50 represents the smallest resolvable object determined byoptical characteristics within the image transfer medium 30 and isdescribed in more detail below. A scaled receptor 54, depicted in frontof the field of view 34 for exemplary purposes, and having a sizedetermined according to the pitch of the sensor 20, is matched or scaledto be about the same size in the object field of view 34 as thediffraction limited point 50 which is a function of the resolvablecharacteristics of the image transfer medium 30.

In other words, the size of any given receptor at the sensor 20 iseffectively reduced in size via the image transfer medium 30 to be aboutthe same size (or matched in size) to the size of the diffractionlimited point 50. This also has the effect of filling the object fieldof view 34 with substantially all of the receptors of the sensor 20, therespective receptors being suitably scaled to be similar in size to thediffraction limited point 50. As will be described in more detail below,the matching/mapping of sensor characteristics to the smallestresolvable object or point within the object field of view 34 definesthe imaging system 10 in terms of absolute spatial resolution and thus,enhances the operating performance of the system.

An illumination source 60 can be provided with the present invention inorder that photons from the source can be transmitted through and/orreflected from objects in the field of view 34 to enable activation ofthe receptors in the sensor 20. It is noted that the present inventioncan potentially be employed without an illumination source 60 ifpotential self-luminous objects (e.g., fluorescent or phosphorescentbiological or organic material sample, metallurgical, mineral, and/orother inorganic material and so forth) emit enough radiation to activatethe sensor 60. Light Emitting Diodes, however, provide an effectiveillumination source 60 in accordance with the present invention.Substantially any illumination source 60 can be applied includingcoherent and non-coherent sources, visible and non-visible wavelengths.However, for non-visible wavelength sources, the sensor 20 and ifnecessary, the optical media of the image transfer medium 30 would alsobe suitably adapted. For example, for an infrared or ultraviolet source,an infrared or ultraviolet sensor 20 and IR or UV suitable opticalcomponents in the image transfer medium 30 would be employed,respectively. Other illumination sources 60 can includewavelength-specific lighting, broad-band lighting, continuous lighting,strobed lighting, Kohler illumination, Abbe illumination, phase-contrastillumination, darkfield illumination, brightfield illumination, and Epiillumination. Transmissive or reflective lighting techniques (e.g.,specular and diffuse) can also be applied.

Reference numeral 80 depicts the outline of an image transfer medium,associated sensor, and computer system which receives image data fromthe associated sensor for generating images in accordance with thepresent invention. It is to be appreciated that these components can beconfigured in a plurality of different combinations such as in variousportable configurations (e.g., hand held or laptop device), stand-aloneconfigurations (e.g., industrial analyzer), or a combination of portableand stand-alone configurations/applications. For example, theseconfigurations can include a plurality of portable imaging systems thatmay be powered by portable power sources, generators, batteries, solar,fuel-cell, other power sources which offer power appropriate to both theimaging system and the associated computer and display system, and canbe distributed throughout differing regions to support various remoteimaging applications. Such applications can include remote medicine orremote industrial applications whereby images are generated in one ormore locations and transmitted to another location or location foranalysis (e.g., remote pathology application, semiconductor qualitycontrol application).

Referring now to FIG. 2, a system 100 illustrates an image transfermedium in accordance with an aspect of the present invention. The imagetransfer medium 30 depicted in FIG. 1 can be provided according to thek-space design concepts described above and more particularly via ak-space filter 110 adapted, configured and/or selected to promote a bandof predetermined k-space frequencies 114 and to mitigate frequenciesoutside of this band. This is achieved by determining a pitch “P”—whichis the distance between adjacent receptors 116 in a sensor (not shown)and sizing optical media within the filter 110 such that the pitch “P”of the receptors 116 is matched in size with a diffraction-limited spot120. The diffraction-limited spot 120 can be determined from the opticalcharacteristics of the media in the filter 110. For example, theNumerical Aperture of an optical medium such as a lens defines thesmallest object or spot that can be resolved by the lens. The filter 110performs a k-space transformation such that the size of the pitch iseffectively matched, “unit-mapped”, projected, correlated, and/orreduced to the size or scale of the diffraction limited spot 120.

It is to be appreciated that a plurality of optical configurations canbe provided to achieve the k-space filter 110. One such configurationcan be provided by an aspherical lens 124 adapted such to perform thek-space transformation and reduction from sensor space to object space.Yet another configuration can be provided by a multiple lens arrangement128, wherein the lens combination is selected to provide the filteringand scaling. Still yet another configuration can employ a fiber optictaper 132 or image conduit, wherein multiple optical fibers or array offibers are configured in a funnel-shape to perform the mapping of thesensor to the object field of view. It is noted that the fiber optictaper 132 is generally in physical contact between the sensor and theobject under examination (e.g., contact with microscope slide). Anotherpossible k-space filter 110 arrangement employs a holographic (or otherdiffractive or phase structure) optical element 136, wherein asubstantially flat optical surface is configured via a hologram (orother diffractive or phase structure) (e.g., computer-generated,optically generated, and/or other method) to provide the mapping inaccordance with the present invention.

The k-space optical design as enabled by the k-space filter 110 is basedupon the “effective projected pixel-pitch” of the sensor, which is afigure derived from following (“projecting”) the physical size of thesensor array elements back through the optical system to the objectplane. In this manner, conjugate planes and optical transform spaces arematched to the Nyquist cut-off of the effective receptor or pixel size.This maximizes the effective resolved image magnification and the FieldOf View as well as the Depth Of Field and the Absolute SpatialResolution. Thus, a novel application of optical theory is provided thatdoes not rely on conventional geometric optical design parameters ofparaxial ray-tracing which govern conventional optics and imagingcombinations. This can further be described in the following manner.

A Fourier transform of an object and an image is formed (by an opticalsystem) in k-space (also referred to as “reciprocal-space”). It is thistransform that is operated on for image optimization by the k-spacedesign of the present invention. For example, the optical media employedin the present invention can be designed with standard, relativelynon-expensive “off-the-shelf” components having a configuration whichdefines that the object and image space are “unit-mapped” or“unit-matched” for substantially all image and object fields. A smallBlur-circle or diffraction-limited spot 120 at the object plane isdefined by the design to match the pixels in the image plane (e.g., atthe image sensor of choice) with substantially one-to-one correspondenceand thus the Fourier transforms of pixelated arrays can be matched. Thisimplies that, optically by design, the Blur-circle is scaled to be aboutthe same size as the receptor or pixel pitch. The present invention isdefined such that it constructs an Intrinsic Spatial Filter such as thek-space filter 110. Such a design definition and implementation enablesthe spectral components of both the object and the image in k-space tobe about the same or quantized. This also defines that the ModulationTransfer Function (MTF) (the comparison of contrast to spatialresolution) of the sensor is matched to the MTF of the object Plane.

FIG. 3 illustrates an optical system 200 in accordance with an aspect ofthe present invention. The system 200 includes a sensor 212 having aplurality of receptors or sensor pixels 214. For example, the sensor 212is an M by N array of sensor pixels 214, having M rows and N columns(e.g., 640×480, 512×512, 1280×1024, and so forth), M and N beingintegers respectively. Although a rectangular sensor 212 havinggenerally square pixels is depicted, it is to be understood andappreciated that the sensor can be substantially any shape (e.g.,circular, elliptical, hexagonal, rectangular, and so forth). It is to befurther appreciated that respective pixels 214 within the array can alsobe substantially any shape or size, the pixels in any given array 212being similarly sized and shaped in accordance with an aspect of thepresent invention.

The sensor 212 can be substantially any technology (e.g., digitalsensor, analog sensor, Charge Coupled Device (CCD) sensor, CMOS sensor,Charge Injection Device (CID) sensor, an array sensor, a linear scansensor) including one or more receptors (or pixels) 214. According toone aspect of the present invention, each of the pixels 214 is similarlysized or proportioned and responsive to light (e.g., visible,non-visible) received from the items under examination, as describedherein.

The sensor 212 is associated with a lens network 216, which isconfigured based on performance requirements of the optical system andthe pitch size of sensor 212. The lens network 216 is operative to scale(or project) proportions (e.g., pixels 214) of the sensor 212 at animage plane established by the position of the sensor 212 to an objectfield of view 220 in accordance with an aspect of the present invention.The object field of view 220 is related to the position of an objectplane 222 that includes one or more items (not shown) under examination.

As the sensor 212 receives light from the object field of view 220, thesensor 212 provides an output 226 that can be directed to a local orremote storage such as a memory (not shown) and displayed from thememory via a computer and associated display, for example, withoutsubstantially any intervening digital processing (e.g., straight bit mapfrom sensor memory to display), if desired. It is noted that local orremote signal processing of the image data received from the sensor 212can also occur. For example, the output 226 can be converted toelectronic data packets and transmitted to a remote system over anetwork for further analysis and/or display. Similarly, the output 226can be stored in a local computer memory before being transmitted to asubsequent computing system for further analysis and/or display.

The scaling (or effective projecting) of pixels 214 provided by the lensnetwork 216 is determined by a novel k-space configuration or design inaccordance with an aspect of the present invention. The k-space designof the lens network 216 promotes predetermined k-space frequencies ofinterest and mitigates frequencies outside the predetermined frequencyband. This has the effect of a band-pass filter of the spatialfrequencies within the lens network 216 and notably defines the imagingsystem 200 in terms of resolution rather than magnification. As will bedescribed below, the resolution of the imaging system 200 determined bythe k-space design promotes a plurality of features in a displayed orstored image, such as having high “Effective Resolved Magnification” (afigure of merit described in following), with related high absolutespatial resolution, large depth of field, larger working distances, anda unitary Modulation Transfer Function as well as other features.

In order to determine the k-space frequencies, a “pitch” or spacing 228is determined between adjacent receptors 214 on the sensor 212. Thepitch (e.g., pixel pitch) corresponds to the center-to-center distanceof adjacent receptors, indicated at 228, which is about the size ordiameter of a single receptor when the sensor includes all equally sizedpixels. The pitch 228 defines the Nyquist “cut-off” frequency band ofthe sensor 212. It is this frequency band that is promoted by thek-space design, whereas other frequencies are mitigated. In order toillustrate how scaling is determined in the imaging system 200, a point230 of a desired smallest resolvable spot size is illustrated at theobject plane 222, wherein the point is derived from resolvablecharacteristics of the lens network 216. The point 230, for example, canrepresent the smallest resolvable object determined by opticalcharacteristics of the lens network 216. That is, the lens network isconfigured to have optical characteristics (e.g., magnification,numerical aperture) so that respective pixels 214 are matched or scaledto be about the same size in the object field of view 220 as the desiredminimum resolvable spot size of the point 230. For purposes ofillustration, a scaled receptor 232 is depicted in front of the field ofview 220 as having a size determined according to the pitch 228 of thesensor 212, which is about the same as the point 230.

By way of illustration, the lens network 216 is designed to effectivelyreduce the size of each given receptor (e.g., pixel) 214 at the sensor212 to be about the same size (e.g., matched in size) to the size of thepoint 230, which is typically the minimum spot size resolvable by thesystem 210. It is to be understood and appreciated that the point 230can be selected to a size representing the smallest resolvable objectdetermined by optical characteristics within the lens network 216 asdetermined by diffraction rules (e.g., diffraction limited spot size).The lens network 216 thus can be designed to effectively scale eachpixel 214 of the sensor 212 to any size that is equal to or greater thanthe diffraction limited size. For example, the resolvable spot size canbe selected to provide for any desired image resolution that meets suchcriteria.

After the desired resolution (resolvable spot size) is selected, thelens network 216 is designed to provide the magnification to scale thepixels 214 to the object field of view 220 accordingly. This has theeffect of filling the object field of view 220 with substantially all ofthe receptors of the sensor 212, the respective receptors being suitablyscaled to be similar in size to the point 230, which corresponds to thedesired resolvable spot size. The matching/mapping of sensorcharacteristics to the desired (e.g., smallest) resolvable object orpoint 230 within the object field of view 220 defines the imaging system200 in terms of absolute spatial resolution and enhances the operatingperformance of the system in accordance with an aspect of the presentinvention.

By way of further illustration, in order to provide unit-mappingaccording to this example, assume that the sensor array 212 provides apixel pitch 228 of about 10.0 microns. The lens network 216 includes anobjective lens 234 and a secondary lens 236. For example, the objectivelens 234 can be set at infinite conjugate to the secondary lens 236,with the spacing between the objective and secondary lenses beingflexible. The lenses 234 and 236 are related to each other so as toachieve a reduction from sensor space defined at the sensor array 220 toobject space defined at the object plane 222. It is noted thatsubstantially all of the pixels 214 are projected into the object fieldof view 220, which is defined by the objective lens 234. For example,the respective pixels 214 are scaled through the objective lens 234 toabout the dimensions of the desired minimum resolvable spot size. Inthis example, the desired resolution at the image plane 222 is onemicron. Thus, a magnification of ten times is operative to back projecta ten micron pixel to the object plane 222 and reduce it to a size ofone micron.

The reduction in size of the array 212 and associated pixels 214 can beachieved by selecting the transfer lens 236 to have a focal length “D2”(from the array 212 to the transfer lens 236) of about 150 millimetersand by selecting the objective lens to have a focal length “D1” (fromthe objective lens 236 to the object plane 222) of about 15 millimeters,for example. In this manner, the pixels 214 are effectively reduced insize to about 1.0 micron per pixel, thus matching the size of the of thedesired resolvable spot 230 and filling the object field of view 220with a “virtually-reduced” array of pixels it is to be understood andappreciated that other arrangements of one or more lenses can beemployed to provide the desired scaling. In view of the foregoingdescription, those skilled in the art will understand and appreciatethat the optical media (e.g., lens network 216) can be designed, inaccordance with an aspect of the present invention, with standard,relatively inexpensive “off-the-shelf” components having a configurationthat defines that the object and image space are “unit-mapped” or“unit-matched” for substantially all image and object fields. The lensnetwork 216 and, in particular the objective lens 234, performs aFourier transform of an object and an image in k-space (also referred toas “reciprocal-space”). It is this transform that is operated on forimage optimization by the k-space design of the present invention.

A small Blur-circle or Airy disk at the object plane is defined by thedesign to match the pixels in the image plane (e.g., at the image sensorof choice) with substantially one-to-one correspondence with the Airydisk and thus the Fourier transforms of pixilated arrays can be matched.This implies that, optically by design, the Airy disk is scaled throughthe lens network 216 to be about the same size as the receptor or pixelpitch. As mentioned above, the lens network 216 is defined so as toconstruct an Intrinsic Spatial Filter (e.g., a k-space filter). Such adesign definition and implementation enables the spectral components ofboth the object and the image in k-space to be about the same orquantized. This also defines that a Modulation Transfer Function (MTF)(the comparison of contrast to spatial resolution) of the sensor can bematched to the MTF of the object Plane in accordance with an aspect ofthe present invention.

As illustrated in FIG. 3, k-space is defined as the region between theobjective lens 234 and the secondary lens 236. It is to be appreciatedthat substantially any optical media, lens type and/or lens combinationthat reduces, maps and/or projects the sensor array 212 to the objectfield of view 220 in accordance with unit or k-space mapping asdescribed herein is within the scope of the present invention.

To illustrate the novelty of the exemplary lens/sensor combinationdepicted in FIG. 3, it is noted that conventional objective lenses,sized according to conventional geometric paraxial ray techniques, aregenerally sized according to the magnification, Numeric Aperture, focallength and other parameters provided by the objective. Thus, theobjective lens would be sized with a greater focal length thansubsequent lenses that approach or are closer to the sensor (or eyepiecein conventional microscope) in order to provide magnification of smallobjects. This can result in magnification of the small objects at theobject plane being projected as a magnified image of the objects across“portions” of the sensor and results in known detail blur (e.g.,Rayleigh diffraction and other limitations in the optics), emptymagnification problems, and Nyquist aliasing among other problems at thesensor. The k-space design of the present invention operates in analternative manner to conventional geometrical paraxial ray designprinciples. That is, the objective lens 234 and the secondary lens 236operate to provide a reduction in size of the sensor array 212 to theobject field of view 220, as demonstrated by the relationship of thelenses.

An illumination source 240 can be provided with the present invention inorder that photons from that source can be transmitted through and/orreflected from objects in the field of view 234 to enable activation ofthe receptors in the sensor 212. It is noted that the present inventioncan potentially be employed without an illumination source 240 ifpotential self-luminous objects (e.g., objects or specimens withemissive characteristics as previously described) emit enough radiationto activate the sensor 212. Substantially any illumination source 240can be applied including coherent and non-coherent sources, visible andnon-visible wavelengths. However, for non-visible wavelength sources,the sensor 212 would also be suitably adapted. For example, for aninfrared or ultraviolet source, an infrared or ultraviolet sensor 212would be employed, respectively. Other suitable illumination sources 240can include wavelength-specific lighting, broad-band lighting,continuous lighting, strobed lighting, Kohler illumination, Abbeillumination, phase-contrast illumination, darkfield illumination,brightfield illumination, Epi illumination, and the like. Transmissiveor reflective (e.g., specular and diffuse) lighting techniques can alsobe applied.

FIG. 4 illustrates a graph 300 of mapping characteristics and comparisonbetween projected pixel size on the X-axis and diffraction-limited spotresolution size “R” on the Y-axis. An apex 310 of the graph 300corresponds to unit mapping between projected pixel size and thediffraction limited spot size, which represents an optimum relationshipbetween a lens network and a sensor in accordance with the presentinvention.

It is to be appreciated that the objective lens 234 (FIG. 3) shouldgenerally not be selected such that the diffraction-limited size “R” ofthe smallest resolvable objects are smaller than a projected pixel size.If so, “economic waste” can occur wherein more precise information islost (e.g., selecting an object lens more expensive than required, suchas having a higher numerical aperture). This is illustrated to the rightof a dividing line 320 at reference 330 depicting a projected pixel 340larger that two smaller diffraction spots 350. In contrast, where anobjective is selected with diffraction-limited performance larger thanthe projected pixel size, blurring and empty magnification can occur.This is illustrated to the left of line 320 at reference numeral 360,wherein a projected pixel 370 is smaller than a diffraction-limitedobject 380. It is to be appreciated, however, that even if substantiallyone-to-one correspondence is not achieved between projected pixel sizeand the diffraction-limited spot, a system can be configured with lessthan optimum matching (e.g., 0.1%, 1%, 2%, 5%, 20%, 95% down from theapex 310 on the graph 300 to the left or right of the line 320) andstill provide suitable performance in accordance with an aspect of thepresent invention. Thus, less than optimal matching is intended to fallwithin the spirit and the scope of present invention.

It is further to be appreciated that the diameter of the lenses in thesystem as illustrated in FIG. 3, for example, should be sized such thatwhen a Fourier Transform is performed from object space to sensor space,spatial frequencies of interest that are in the band pass regiondescribed above (e.g., frequencies utilized to define the size and shapeof a pixel) are substantially not attenuated. This generally impliesthat larger diameter lenses (e.g., about 10 to 100 millimeters) shouldbe selected to mitigate attenuation of the spatial frequencies ofinterest.

Referring now to FIG. 5, a Modulation Transfer function 400 isillustrated in accordance with the present invention. On a Y-axis,modulation percentage from 0 to 100% is illustrated defining percentageof contrast between black and white. On an X-axis, Absolution SpatialResolution is illustrated in terms of microns of separation. A line 410illustrates that modulation percentage remains substantially constant atabout 100% over varying degrees of spatial resolution. Thus, theModulation Transfer Function is about 1 for the present invention up toabout a limit imposed by the signal to noise sensitivity of the sensor.For illustrative purposes, a conventional optics design ModulationTransfer Function is illustrated by line 420 which may be an exponentialcurve with generally asymptotic limits characterized by generallydecreasing spatial resolution with decreasing modulation percentage(contrast).

FIG. 6 illustrates a quantifiable Figure of Merit (FOM) for the presentinvention defined as dependent on two primary factors: Absolute SpatialResolution (R_(A), in microns), depicted on the Y axis and the Field OfView (F, in microns) depicted on the X axis of a graph 500. A reasonableFOM called “Spatial Field Number” (S), can be expressed as the ratio ofthese two previous quantities, with higher values of S being desirablefor imaging as follows:S=F/R _(A)

A line 510 illustrates that the FOM remains substantially constantacross the field of view and over different values of absolute spatialresolution which is an enhancement over conventional systems.

FIGS. 7, 8, 14, 15, 16, and 20 illustrate methodologies to facilitateimaging performance in accordance with the present invention. While, forpurposes of simplicity of explanation, the methodologies may be shownand described as a series of acts, it is to be understood andappreciated that the present invention is not limited by the order ofacts, as some acts may, in accordance with the present invention, occurin different orders and/or concurrently with other acts from that shownand described herein. For example, those skilled in the art willunderstand and appreciate that a methodology could alternatively berepresented as a series of interrelated states or events, such as in astate diagram. Moreover, not all illustrated acts may be required toimplement a methodology in accordance with the present invention.

Turning now to FIG. 7 and proceeding to 610, lenses are selected havingdiffraction-limited characteristics at about the same size of a pixel inorder to provide unit-mapping and optimization of the k-space design. At614, lens characteristics are also selected to mitigate reduction ofspatial frequencies within k-space. As described above, this generallyimplies that larger diameter optics are selected in order to mitigateattenuation of desired k-space frequencies of interest. At 618, a lensconfiguration is selected such that pixels, having a pitch “P”, at theimage plane defined by the position of a sensor are scaled according tothe pitch to an object field of view at about the size of adiffraction-limited spot (e.g., unit-mapped) within the object field ofview. At 622, an image is generated by outputting data from a sensor forreal-time monitoring and/or storing the data in memory for directdisplay to a computer display and/or subsequent local or remote imageprocessing and/or analysis within the memory.

FIG. 8 illustrates a methodology that can be employed to design anoptical/imaging system in accordance with an aspect of the presentinvention. The methodology begins at 700 in which a suitable sensorarray is chosen for the system. The sensor array includes a matrix ofreceptor pixels having a known pitch size, usually defined by themanufacturer. The sensor can be substantially any shape (e.g.,rectangular, circular, square, triangular, and so forth). By way ofillustration, assume that a sensor of 640×480 pixels having a pitch sizeof 10 μm is chosen. It is to be understood and appreciated that anoptical system can be designed for any type and/or size of sensor arrayin accordance with an aspect of the present invention.

Next at 710, an image resolution is defined. The image resolutioncorresponds to the smallest desired resolvable spot size at the imageplane. The image resolution can be defined based on the application(s)for which the optical system is being designed, such as any resolutionthat is greater than or equal to a smallest diffraction limited size.Thus, it is to be appreciated that resolution becomes a selectabledesign parameter that can be tailored to provide desired imageresolution for virtually any type of application. In contrast, mostconventional systems tend to limit resolution according to Rayleighdiffraction, which provides that intrinsic spatial resolution of thelenses cannot exceed limits of diffraction for a given wavelength.

After selecting a desired resolution (710), a suitable amount ofmagnification is determined at 720 to achieve such resolution. Forexample, the magnification is functionally related to the pixel pitch ofthe sensor array and the smallest resolvable spot size. Themagnification (M) can be expressed as follows: $\begin{matrix}{M = \frac{x}{y}} & {{Eq}.\quad 1}\end{matrix}$wherein:

-   -   x is the pixel pitch of the sensor array; and    -   y is the desired image resolution (minimum spot size).

So, for the above example where the pixel pitch is 10 μm and assuming adesired image resolution of 1 μm, Eq. 1 provides an optical system ofpower ten. That is, the lens system is configured to back-project each10 μm pixel to the object plane and reduce respective pixels to theresolvable spot size of 1 micron.

The methodology of FIG. 8 also includes a determination of a NumericalAperture at 730. The Numerical Aperture (NA) is determined according towell-established diffraction rules that relate NA of the objective lensto the minimum resolvable spot size determined at 710 for the opticalsystem. By way of example, the calculation of NA can be based on thefollowing equation: $\begin{matrix}{{NA} = \frac{0.5 \times \lambda}{y}} & {{Eq}.\quad 2}\end{matrix}$where:

-   -   λ is the wavelength of light being used in the optical system;        and    -   y is the minimum spot size (e.g., determined at 710).

Continuing with the example in which the optical system has a resolvedspot size of y=1 micron, and assuming a wavelength of about 500 nm(e.g., green light), a NA=0.25 satisfies Eq. 2. It is noted thatrelatively inexpensive commercially available objectives of power 10provide numerical apertures of 0.25.

It is to be understood and appreciated that the relationship between NA,wavelength and resolution represented by Eq. 2 can be expressed indifferent ways according to various factors that account for thebehavior of objectives and condensers. Thus, the determination at 730,in accordance with an aspect of the present invention, is not limited toany particular equation but instead simply obeys known general physicallaws in which NA is functionally related to the wavelength andresolution. After the lens parameters have been designed according tothe selected sensor (700), the corresponding optical components can bearranged to provide an optical system (740) in accordance with an aspectof the present invention.

Assume, for purposes of illustration, that the example optical systemcreated according to the methodology of FIG. 8 is to be employed formicroscopic-digital imaging. By way of comparison, in classicalmicroscopy, in order to image and resolve structures of a sizeapproaching 1 micron (and below), magnifications of many hundredsusually are required. The basic reason for this is that such opticsconventionally have been designed for the situation when the sensor ofchoice is the human eye. In contrast, the methodology of FIG. 8 designsthe optical system in view of the sensor, which affords significantperformance increases at reduced cost.

In the k-space design methodology, according to an aspect of the presentinvention, the optical system is designed around a discrete sensor thathas known fixed dimensions. As a result, the methodology can provide afar more straight-forward, robust, and inexpensive optical system designapproach to “back-project” the sensor size onto the object plane andcalculate a magnification factor. A second part of the methodologyfacilitates that the optics that provide the magnification have asufficient NA to optically resolve a spot of similar dimensions as theback-projected pixel. Advantageously, an optical system designed inaccordance with an aspect of the present invention can utilize customand/or off-the-shelf components. Thus, for this example, inexpensiveoptics can be employed in accordance with an aspect of the presentinvention to obtain suitable results, but well-corrected microscopeoptics are relatively inexpensive. If custom-designed optics areutilized, in accordance with an aspect of the present invention, thenthe range of permissible magnifications and numerical apertures becomessubstantial, and some performance gains can be realized over the use ofoff-the-shelf optical components.

In view of the concepts described above in relation to FIGS. 1-8, aplurality of related imaging applications can be enabled and enhanced bythe present invention. For example, these applications can include butare not limited to imaging, control, inspection, microscopy and/or otherautomated analysis such as:

-   -   (1) Bio-medical analysis (e.g., cell colony counting, histology,        frozen sections, cellular cytology, Meachanical, Laser or        radiation-based, and other Micro-dissection, Haematology,        pathology, oncology, fluorescence, interference, phase and many        other clinical microscopy applications);    -   (2) Particle Sizing Applications (e.g., Pharmaceutical        manufacturers, paint manufacturers, cosmetics manufacturers,        food process engineering, and others);    -   (3) Air quality monitoring and airborne particulate measurement        (e.g., clean room certification, environmental certification,        and so forth);    -   (4) Optical defect analysis, and other requirements for high        resolution microscopic inspection of both transmissive and        opaque materials (as in metallurgy, automated semiconductor        inspection and analysis, automated vision systems, 3-D imaging        and so forth); and    -   (5) Imaging technologies such as cameras, copiers, FAX machines        and medical systems as well as other technologies/applications        which are described in more detail below.

FIGS. 9-16 illustrate possible example systems that can be constructedemploying the concepts previously described above in relation to FIGS.1-8. It is to be appreciated that the pixel-mapping components andconcepts can be applied to two-dimensional imaging systems and can alsobe applied to multi-dimensional system as well. For instance, more thanone image can be taken using the subject imaging system, whereinrespective images may be combined with each other to form highlyresolved three-dimensional images using image reconstruction techniques.

FIG. 9 depicts an example packaging concept and system 900 for animaging system adapted in accordance with the present invention. In thisaspect, the system 900 includes imaging components such as a sensors,optics, and adjustments that provide imaging data to an associatedcomputer (not shown) through substantially any desired couplingtechnique such as a Firewire, USB port, parallel port, infrared, and soforth. It is to be appreciated however, that a computer system (orportions thereof, e.g., memory components) could be provided within thesystem 900.

The system 900 can include such aspects as a k-space access panel at910, various adjustment devices 920 and 930 for adjusting or positioninglenses and/or stages for viewing desired objects, and one or more accessspaces 934 to insert an object sample. These adjustments, as well assample ingress, egress, and handling, can be automated such as via servocontrols, manual controls, or combinations of manual and automaticcontrols. Other features of the system 900 include Koehler iris controls940, a projection/condensor access panel 944, and a transmissiveillumination module 950 having an associated transmission illuminationiris control 954. Other controls include an objective turret dialcontrol 960 and an EPI-illumination control 964 associated with anEpi-illumination module 970. An Epi-Illumination access space 980 canalso be provided.

FIG. 10 depicts a system assembly layout 1000 for the system 900illustrated in FIG. 9, wherein such system can be configured in a handheld or portable arrangement (e.g., 10 inches by 12 inches apparentfootprint). At 1010, an LED solid-state illumination source having anassociated holographic/diffractive optical element is provided. At 1020,a projection condensor module is provided. At 1030, an illuminationmodule assembly is provided. At 1040, a Koehler/Abbe microscopecondenser and stage assembly is provided, each of the which may haveaxially moveable components for object focus. At 1050, turret mountedobjective lenses are provided that can be axially moveable and lockablefor object field of view focus. It is to be appreciated that suchmultiple objective configurations may include as many or few lenses asis practicable or desirable in any chosen design configuration. Also,rather than a turret, a linear slide configuration of lenses can beprovided.

At 1060, a k-space access section is provided. At 1070, an optical pathfor filters and/or other optical components is provided. At 1078, anEpi-illumination assembly is provided having an associatedholographic/diffractive optical element. It is noted that any of thelight sources described herein can be adapted or changed for variousdifferent wavelengths. Also, different types of sources can be provided.For example, in certain low power applications, a conventional LED(having differing wavelengths) can be employed, whereas for otherapplications, a light source such as a Luxeon star can be employed. At1080 a sensor detector module and access entry is provided. At 1084, adetector connector for sending image data to a computer is provided. At1090, an exemplary battery/power module is provided and an AC or otherpower input can be provided at 1094.

FIG. 11 illustrates an optical train assembly 1100 in accordance with anaspect of the present invention. The assembly 1100 includes one or moreof the following: an illumination source 1110, a holographic/diffractiveoptical element 1114, a condensor projection lens 1120 (generallyaspheric), an iris diaphragm or aperture 1124, a field lens 1130, aKohler or Abbe microscope condenser 1034, a moveable stage assembly1140, a sample 1144 (e.g., transparent or opaque sample or slide),turret mounted microscope objectives 1150 (axially moveable and lockablefor object Field of View focus), a beam splitter 1154, a telan or tubeor field lens 1160, a detector system 1164 (CMOS, CCD, and so forth), anEPI illumination assembly 1168 having a projection lens 1170, an irisdiaphragm/aperture 1174, a holographic/diffractive optical element 1180,and an illumination source 1184 (wavelength selectable devices).

FIG. 12 illustrates an alternative portable imaging and microscopedevice 1200 in accordance with an aspect of the present invention. Inthis aspect, a digital camera 1210 is adapted with a mapping lens 1220(or lens assembly) to provide a hand held microscopic imaging device.The mapping lens 1220 provides projected pixel mapping or correlationbetween the camera sensor and a diffraction-limited spot defined by thelens. It is noted that in one example of a medical imager, by usingnumbers (below), approximately 1.75×1.32 millimeters of object can beimaged by the camera 1210—yielding approximately 25 pixels per red cell,and 100-500 pixels per white cell, at a resolution of 1 micron, forexample. A haematology analysis can proceed with 1,000 cells (total),thus a single image will be capable of holding sufficient informationfor a full cell count. In an example design the following may apply:

-   -   1. select a desired resolution, e.g., 1 micron.    -   2. calculate that the objective lens would need an NA of        approximately 0.3 based on example resolution of 1 micron.    -   3. calculate that the magnification required to get from 1        micron of resolution to 5.4 microns of pixel-quadrad pitch is        approximately 5×    -   4. construct a k-space-transfer microscope with a 5×NA=0.3        objective lens    -   5. employ a digital camera (e.g., Sony DSC-F828), remove the        lens sold with camera, and mate the camera to the k-space        microscope described in 4.

In general, “desired” is what a CCD sensor (or other type) is capableof, wherein “3-filter” is what current CCDs perform (usually RGGBquadrads of pixels) and “4-filter” such as Sony performs (RGBC quadrad).Other non-rectangular geometries can also be employed, such as hexagonalor pentagonal, whereby more filters can be adapted on the extra pixelsto further fill-in the colors currently missed by a 3-filterarrangement.

FIGS. 13-16 describe another exemplary portable system in accordancewith the present invention. In general, in nearly all cases whereby adigital sensor is attached to an imaging system capable of higheffective magnification, the resulting system is non-portable due toconstraints of size, required power, and support infrastructure. One ofthe main considerations is that conventional systems are designed forhuman use, and do not take advantage of the inherent possibilities of adiscrete sensor approach.

When an imaging system is designed to take full advantage of theproperties of a discrete sensor array, many of the design parametersthat have to be considered for a conventional system can be discarded,leading to a greatly simplified system which exhibits a similar (orbetter) effective magnification, similar (or better) resolution, andmuch lower light requirements.

As an example of a reduced-size system, the following describes afully-integrated haematology system (or other application) which hasdirect relevance to emerging (third-world) countries where a simple,fast blood analysis (e.g., HIV, sickle-cell anaemia) would be of greatvalue.

Some considerations are that the system should be capable of beingpowered from a variety of sources (battery, line voltage, solar cell),should have a minimum of moving parts, and should have robust software(i.e., embedded firmware). In addition, the system cost should beminimal. Also, the system should be capable of performing all commonlyutilized tests that involve samples as presented to a microscope on aslide substrate. The following describes some possible considerations:

-   -   sensor considerations:        -   the sensor should be heavily integrated and not require            complex support circuitry        -   the sensor should require very little power        -   the sensor should be sensitive to low levels of light        -   the sensor should have a wide dynamic range        -   the sensor should have a large number of active sites            (pixels) but not be so large as to require excessive data            transfer times (i.e., 1 k×1 k array)    -   optical considerations:        -   the system should conform to k-space design parameters        -   the system should have as wide a field of view as practical        -   the system should be as near to focus-free as practical        -   the system should have a large depth of field        -   the system should use simple optical components    -   lighting considerations:        -   should have low power requirements        -   should require minimal user adjustment        -   should have extended life    -   mechanical considerations        -   the system should be very robust to allow for field-usage        -   the system should allow easy field-servicing        -   the system should be as modular as possible        -   the system should be light to allow for portability    -   digital considerations        -   computing power is not a necessity, so a moderately            low-performance computing platform will suffice        -   very low electrical power consumption        -   common computing functions should be integrated onto the            main computing platform        -   outputs for LCD and CRT should be provided for flexibility        -   provision for external storage for any presently extant or            future strorage media should be made, with interfaces for            floppy disk, memory stick, USB memory adapter, printer,            CDROM, DVDROM, and/or hard disk        -   basic computing functions, such as the operating system and            application software should be embedded in non-volatile            storage such as ROM or flash memory, leading to diskless            operation        -   the OS and application software should be robust and capable            of extension either on-site by the user, or remotely by            e.g., dialup or wireless internet connection.

One example implementation of the above system:

-   -   sensor:        -   a CMOS array with the above-described characteristics, and            is available in sizes from 640×480 to 1280×10²⁴ with pixels            of approximately 5 microns.        -   typical power requirements are in the low 10's of            milliwatts.    -   optics as depicted in the optical configuration 1300 of FIG. 13:        -   for a haematology analysis system, a base resolution of one            micron per pixel is quite sufficient to resolve and identify            red cells (5 um) and white cells (25 um).        -   referring to the proposed CMOS sensor, this leads to an            optical magnification of 5× requirement to map a single            pixel onto the object plane with a resolution of 1 um.        -   for a magnification of 5×, and a projected pixel-pitch of 1            um, the required Numerical Aperture of the imaging lens to            provide a diffraction-limited 1 um spot is 0.25.        -   for such modest magnification requirements, the optical            design can be met using a simple Gauss-pair of achromats            1310 and 1320. The optimal design would use            -   a sensor lens of focus f=150 mm, diameter d=25 mm            -   an imaging lens of f=30 mm, d=25 mm (giving an NA of                slightly greater than 0.25)        -   referring to an example 1280×10²⁴ pixel sensor and the above            optical prescription, an imaging system is provided capable            of 1 um resolution with a field of view of 1.2 mm×1 mm, and            depth of focus (field) approaching 10 microns.

FIG. 14 illustrates a lighting system 1400 having possible parameters asfollows:

-   -   white LEDs should be used with consequently low power        requirements (<50 mW) and long operating life (50,000+hours)    -   light homogenisation via holographic diffusers should be used        (leading to a virtual point-source)    -   Kohler sub-stage (transmissive) lighting should be provided    -   option should be made for white-light or UV epi-illumination if        required.

-   optomechanics    -   components should be standard sizes (e.g. 25 mm/1 inch diameter)    -   components should be corrosion-resistant (due to the        commonly-met chemical environment in haematology)    -   all components should be easily assembled in the form of modules    -   e.g., components from Thorlabs, Linos and similar

FIG. 15 illustrates an example processing system 1500 having possibleconfigurations of:

-   -   the processor of can be an ARM range (Advanced RISC Machines or        other type), which are widely accepted as having the best        performance to power ratio. Additionally, many of the ARM        variants are available as “system on chip” designs, where a CPU,        FPU, IO and Video subsystems are fully integrated into a        single-chip package (e.g., ARM 7500 FE, StrongARM 1110)    -   the operating system can be a RISCOS (or other type), which is a        ROM-based OS that has both high- and low-level abstractions, is        very robust, and has been deployed since 1987. It also has a        very simple and intuitive graphical user interface. Code        density, due to the RISC techniques employed, is very good, and        major applications generally only use 100's of kilobytes.    -   typically, complete computing systems can be obtained that make        use of RISCOS and the ARM processors in the form of single-board        computers. Power requirements very rarely exceed 5-10 Watts.        On-board storage is typically supplied in the form of flash        memory (16-64 megabytes). Common sizes for the computer boards        are 100 mm×150 mm, and this has lead to the availability of        low-power LCD displays with integrated computer boards (e.g.,        Explan SOLO, Castle SLYM)    -   for a completely integrated haematology solution (or other        application), direct interfacing of the sensor array into the        main memory space of the host computer avoids complications and        expense consequent with standard interfaces (e.g., USB,        IEEE-1394), and also allows simple control of the sensor        functions. Some sophistication can be achieved by using        double-buffered memory (e.g., VRAM) to act as an arbitrator        between the sensor and the host CPU.    -   for the above-described system, power requirements generally        fall within a 10 watt power budget, and can be met by battery,        line, or solar-cell supplies.        Software:

-   should be implemented with a simple graphical user interface

-   should be capable of identifying red cells, white cells, and    sickle-cells (deformed erithrocytes)

-   should be capable of performing simple counts and blood    differentials

-   should present results visually (displayed image) and textually    (data list)

-   should offer off-line storage on removable media (e.g., floppy disk,    memory stick) or as a simple printout

-   should be capable of being expanded via a modular interface to allow    for more sophisticated analyses

-   should be capable of being distributed in varying formats (e.g., on    a storage medium, as a data stream via remote connection, or as a    ROM-module).

-   should be well-documented and open-source, if possible, to allow for    onsite modifications

FIG. 16 illustrates a system 1600 that provides the following features:

-   -   fully-integrated and portable    -   a baseplate 1610 holding an optics/sensor module, a lighting        module, and a sample presentation module    -   single-board computer coupled to baseplate 1610, carrying        display (LCD)    -   expansion unit carrying printer/floppy disk.

FIG. 17 illustrates an alternative imaging design in accordance with anaspect of the present invention. In this aspect, a highly modular andportable system 1700 is illustrated from a top view. The system 1700includes a handheld computer 1710 having a digital camera integrated orassociated therewith. Such computers 1710 can include wired ports suchas USB ports or wireless ports such as Bluetooth, for example, fortransferring electronic images between locations in accordance with thepresent invention. Although, a Hewlett-Packard iPAQ computer isillustrated at 1710, it is to be appreciated that a plurality of vendorssupply such devices such as Sony, Casio, Palm, Dell, Viewsonic, Toshiba,Sharp, Phillips, JVC, and so forth, for example.

Other components illustrated on the system 1700 for generatingmicroscopic images can include one or more mirrors at 1720 and 1730, amatching lens at 1740, a resolution lens at 1750, a stage component at1760 where the object plane resides, an illumination lens at 1770, alight source mount at 1780 (e.g., LED mount), and a power supply 1790(e.g., battery, regulated AC supply). In this example, the lens 1740 and1750 map the characteristics of a pixel associated with the camera thathas been integrated with the computer 1710 in accordance with thediffraction limits of the lens as described above. For example, if a 2.7micron pixel was the pixel pitch associated with the camera, and a 0.65Numerical Aperture resolution lens 1750 were selected providing about384 nanometer resolution for white light, then the overall magnificationof the matching lens 1740 and the resolution lens 1750 would be selectedto be about 7.03× (x refers to times magnified). Thus, 7.03× in thisexample maps 384 nanometer resolution with 2.7 micron pixels (2.7/0.384is approximately 7.03125). Although components are laid out in a planaror two-dimensional arrangement in the system 1700, it is to beappreciated that the components can be arranged in two and/orthree-dimensional arrangement, wherein optics, computers, components,and/or sensors are configured circumferentially or other manner withrespect to one another in order to minimize planar real estate of theoverall configuration. Other views of the system 1700 are illustrated at1792 through 1798.

It is noted that many manufacturers and other institutions teach that toconform to the Nyquist criterion that at least two and preferably threepixels should be provided for each diffraction-limited spot or parameterdefined by the Numerical Aperture of the lens. This is substantially incontravention to the present invention that maps less than two pixelsper spot (e.g., the size of one pixel matched via the optics to the sizeof the diffraction-limited spot). Thus, the camera described abovegenerally includes a sensor having one or more pixels, whereby thepixels have a pixel pitch or resolution parameter, and the pixel pitchis correlated to the optical components. Thus, in accordance with thepresent invention, any mapping that is less than two pixels per spot iswithin the scope of the present invention, wherein it is noted thatfractional mappings are possible (e.g., 1.999 pixels per spot, 1.5pixels per spot, 1.0 pixels per spot, 0.9 pixels per spot, 0.7 pixelsper spot).

In one aspect of the invention, the pixel size is one of a pixel pitch(for any pixels), pixel length (for square pixels), and pixel width (forrectangular pixels), that is about 0.1 microns or more and about 20microns or less. In another aspect of the invention, the pixel size isone of a pixel pitch (for any pixels), pixel length (for square pixels),and pixel width (for rectangular pixels), that is about 0.25 microns ormore and about 15 microns or less. In yet another aspect of theinvention, the pixel size is one of a pixel pitch (for any pixels),pixel length (for square or rectangular pixels), and pixel width (forsquare or rectangular pixels), that is about 0.5 microns or more andabout 10 microns or less. In still yet another aspect of the invention,the pixel size is one of a pixel pitch (for any pixels), pixel length(for square or rectangular pixels), and pixel width (for square orrectangular pixels), that is about 4 microns or more and about 9 micronsor less.

In one aspect of the invention, the ratio of the projected pixel size tothe diffraction spot size, both in the object plane and determined bypitch, length, width, or diameter, is between 1:2 and 2:1. In anotheraspect of the invention, the ratio of the projected pixel size to thediffraction spot size, both in the object plane and determined by pitch,length, width, or diameter, is from about 1:1.9 to about 1.9:1. Inanother aspect of the invention, the ratio of the projected pixel sizeto the diffraction spot size, both in the object plane and determined bypitch, length, width, or diameter, is from about 1:1.7 to about 1.7:1.In another aspect of the invention, the ratio of the projected pixelsize to the diffraction spot size, both in the object plane anddetermined by pitch, length, width, or diameter, is from about 1:1.5 toabout 1.5:1. In another aspect of the invention, the ratio of theprojected pixel size to the diffraction spot size, both in the objectplane and determined by pitch, length, width, or diameter, is from about1:1.4 to about 1.4:1. In another aspect of the invention, the ratio ofthe projected pixel size to the diffraction spot size, both in theobject plane and determined by pitch, length, width, or diameter, isfrom about 1:1.3 to about 1.3:1. In another aspect of the invention, theratio of the projected pixel size to the diffraction spot size, both inthe object plane and determined by pitch, length, width, or diameter, isfrom about 1:1.2 to about 1.2:1. In another aspect of the invention, theratio of the projected pixel size to the diffraction spot size, both inthe object plane and determined by pitch, length, width, or diameter, isfrom about 1:1.1 to about 1.1:1.

With respect to another consideration, the pixel size matching to thediffraction-limited spot results in a tuning of the lenses (or opticalcomponents) and pixels so that desired “spatial” frequencies of interestare received by respective pixels. Without such matching (as inconventional microscopy systems) adjoining pixels may individuallyreceive only a subset of “spatial frequencies” of interest thereby nottaking full advantage of capabilities of respective pixels. Moreover,since the pixels are unaware (e.g., not correlated to) of what subset ofthe spatial frequencies they are respectively receiving, indeterminacyresults when trying to reconstruct the superset of spatial frequenciesassociated with a diffraction-limited spot as received by the set ofadjoining pixels. On the other hand, by size matching individual pixelsto the diffraction-limited spot associated with a given set of lenses asin the present invention, such indeterminacy is substantially mitigatedsince the superset of spatial frequencies of interest are substantiallyreceived by individual pixels. Because there are gaps between pixels inan array and thousands/millions of pixels being exposed to an imageprojection, there may be some level of indeterminacy; however, thesubject invention significantly mitigates such indeterminacy as comparedto conventional systems that fail to contemplate let alone address theadvantages associated with the tuning that results by size mapping ofpixels to the diffraction-limited spot as in the subject invention. Inview of the pixel-mapping concepts described above, the followingconcepts can also be considered:

1. Substantially ensure that all of the information contained in asingle diffraction-limited spot is captured by the pixel (sensor).

2. Provide a significantly enhanced signal-to-noise ratio (SNR) perpixel due to the information in the spot being captured by the pixel.Thus, if two pixels per diffraction-limited spot were provided forexample in a conventional system, the intensity per pixel is reduced toabout a quarter of the about 1:1 matching case of the present invention.

3. There is no guarantee that a diffraction-limited spot (DLS) has auniform distribution of energy, thus in the multiple-pixel case per spotas in conventional systems, one has substantially no concept of whichpixel is sampling which bit of the DLS.

4. Generally, references to the “point spread response” (PSR) refer tothe instrument capturing the image, and not the image itself. Thus, aDLS can be modeled as a convolute of the PSR and the fourier transformof the blob or object specimen being imaged.

To illustrate the above concepts, the following tables are provided tohighlight some example differences between the present invention andconventional microscopic systems. Microscope Comparison ConventionalDesign (Optimized for human eye) Conventional Design Examples: Total(with 10× Depth of Working eyepiece) Resolution NA Field DistanceObjective Magnification 2,500 nm 0.10  100 um 22.0 mm  4×  40× 1,000 nm0.25   16 um 10.5 mm 10× 100×   625 nm 0.40 6.25 um 1.20 mm 20× 200×  384 nm 0.65 2.40 um 0.56 mm 40× 400×   200 nm 1.25 0.64 um 0.15 mm(oil) (oil) 100×  1000× 

By comparison to the above table, if a 2.7 micron pixel were employed,for example, the present invention can provide 2500 nm resolution withabout 1.1× magnification, 1000 nm resolution with about 2.7×magnification, 625 nm resolution with about 4.32× magnification, 384 nmresolution with about 7.03× magnification, and 200 nm resolution withabout 13.5× magnification. Performance Comparison between ConventionalSystems and Present Invention Present invention lens design diameterabout 25 mm, for example. Optimum conventional resolution occurs atapproximately 1000 × NA (See standard optics literature) ConventionalMicroscope Magni- fication Required For Example Present InventionOptical Parameters resolution Resolution Depth Working Magni- (10×Perform- Resolution NA of Field Distance fication eyepiece) ance 2,500nm 0.10  100 um 125 mm  1× 100× 1/100 1,000 nm 0.25   16 um 48 mm 3×250× 1/80    625 nm 0.40 6.25 um 28 mm 4× 400× 1/100   384 nm 0.65 2.40um 14 mm 7× 650× 1/93 

Thus, by observing the above tables, it is illustrated that resolutionin the present invention can be achieved with about 100 times lessmagnification than conventional systems employ when viewed in terms of a2.7 micron pixel. This facilitates such features as greatly improvedworking distances for example along with allowing high performance, lowcost, compact, modular, and robust microscopy systems to be employed.

It is noted that many variants are possible in accordance with thepresent invention. For example, many cell phones are equipped withdigital cameras and thus, the cell phone or similar instrument could beemployed in place of or in addition to the computer 1710 describedabove. Consequently, if an image were captured by the cell phone, theimage could be transferred to various locations by dialling a respectivenumber and transferring the image or images to the location dialled(e.g., web site, remote server, another cell phone or computer). Inanother example, the light source 1780 described above can be variedaccording to differing situations. For example, the light source caninclude a laser light source or other type that is tuned or adjusted(e.g., manually and/or automatically) to different frequencies in orderto fine-tune the characteristics of the diffraction-limited spot inorder to provide desired mapping to the pixel in accordance with thepresent invention. Alternatively, if a lens or sensor were changed andhaving differing characteristics, the light source can be adjustedaccordingly to map the characteristics of the diffraction-limited spotto the characteristics of the projected pixel in the object field ofview.

It is to be appreciated that substantially any system or components thatperforms pixel-mapping or resolution-mapping in an approximatelyone-to-one manner is considered within the scope of the presentinvention. In one example, although pixelated sensor arrays aregenerally employed, other type sensors can be mapped in accordance withthe present invention. For example, a beam scanning type sensor (e.g.,vidicon tube) that is scanned by a beam of light to reproduce an imagecaptured thereon can be described by a beam spot having finite physicaldimensions (e.g., beam diameter) that is moved across the sensor todetect images thereon. In this example, the optics can be adapted suchthat the dimensions of the beam spot are mapped to the dimensions of thebeam diameter or spot in the object field of view as previouslydescribed.

FIG. 18 illustrates an excitation application 1800 in accordance with anaspect of the present invention that can be employed with the systemsand processes previously described. A k-space system is adapted inaccordance with the present invention having a light system thatincludes a light source at 1810, such as a Light Emitting Diode (LED),emitting light having a wavelength of about 250 to about 400 nm (e.g.,ultraviolet light). The LED can be employed to provide forepi-illumination or trans-illumination as described herein (or othertype). The use of such an LED (or other UV light source) also enableswave-guide illumination in which the UV excitation wavelength isintroduced onto a planar surface supporting the object under test at1820, such that evanescent-wave coupling of the UV light can excitefluorophores within the object. For example, the UV light can be (butnot limited to) provided at about a right angle to a substrate on whichthe object lies. At 1830, the LED (or other light source or combinationsthereof) can emit light for a predetermined time period and/or becontrolled in a strobe-like manner emitting pulses at a desired rate.

At 1840, excitation is applied to the object for the period determinedat 1830. At 1850, automated and/or manual analysis is performed on theobject during (and/or thereabout) the excitation period. It is notedthat the excitation application 1800 described herein can apply tovarious processes. In one aspect, this includes a process wherein ahigh-energy photon (short wavelength) is absorbed and subsequentlyre-emitted as a lower-energy photon (longer wavelength). The timeinterval between absorption and emission generally determines whetherthe process is one of fluorescence or phosphorescence—which can also bedefined as a “down-conversion” process.

By way of illustration, the object which is sensitive to ultraviolet inthat it absorbs/emits photons in response to excitation of UV light fromthe light source. Fluorescence (or phosphorescence) is a condition of amaterial (organic or inorganic) in which the material undergoes aprocess wherein a high-energy photon (short wavelength) is absorbed andsubsequently re-emitted as a lower-energy photon (longer wavelength).The time interval between absorption and emission generally determineswhether the process is one of fluorescence or phosphorescence—which canalso be defined as a “down-conversion” process. It can also continue toemit light while absorbing excitation light. Both Fluorescence andphosphorescence can be an inherent property of a material (e.g.,auto-fluorescence) or it can be induced, such as by employingfluorochrome stains or dyes. The dye can have an affinity to aparticular protein, chemical component, physical component, and/or otherreceptiveness so as to facilitate revealing or discovering differentconditions associated with the object. In one particular example,fluorescence microscopy (or phosphorescence microscopy) and/or digitalimaging provide a manner in which to study various materials thatexhibit secondary fluorescence (or phosphorescence).

By way of further example, the UV LED (or other source) can produceintense flashes of UV radiation for a short time period, with an imagebeing constructed by a sensor (sensor adapted to the excitationwavelength) a short time later (e.g., milliseconds to seconds). Thismode can be employed to investigate the time decay characteristics ofthe fluorescent (or phosphorescent) components of the object (or sample)being tested. This may be important where two parts of the object (ordifferent samples) may respond (e.g., fluoresce/phosphorescencesubstantially the same under continuous illumination, but may havediffering emission decay characteristics.

As a result of using the UV light source, such as the LED, the lightfrom the light source can cause at least a portion of the object undertest to emit light, generally not in the ultraviolet wavelength. Becauseat least a portion of the object fluoresces, (or phosphoresces) pre- orpost-fluorescence/phosphorescence images can be correlated relative tothose obtained during fluorescence/phosphorescence of the object toascertain different characteristics of the object. In contrast, mostconventional systems are configured to irradiate a specimen and then toseparate the weaker re-radiating fluorescent light from the brighterexcitation light, typically through filters. In order to enabledetectable fluorescence, such conventional systems usually requirepowerful light sources. For example, the light sources can be mercury orxenon arc (burner) lamps, which produce high-intensity illuminationpowerful enough to image fluorescence specimens. In addition to runninghot (e.g., typically 100-250 Watt lamps), these types of light sourcestypically have short operating lives (e.g., 10-100 hours). In addition,a power supply for such conventional light sources often includes atimer to help track the number of use hours, as arc lamps tend to becomeinefficient and degrade with decreasing or varying illumination outputwith use. The lamps are also more likely to shatter, if utilized beyondtheir rated lifetime. Moreover, conventional light sources such as Xenonand mercury arc lamps (burners) generally do not provide even intensityacross the desired emission spectrum from ultraviolet to infrared, asmuch of the intensity of the mercury burner, for example, is expended inwavelengths across the near ultraviolet. This often requires precisionfiltering to remove undesired light wavelengths. Accordingly, it will beappreciated that using a UV LED, in accordance with an aspect of thepresent invention, provides a substantially even intensity at a desiredUV wavelength to mitigate power consumption and heat generated throughits use. Additionally, the replacement cost of a LED light source issignificantly less than conventional lamps.

In accordance with the foregoing discussion it will be appreciated thatthe excitation source may also be any other light source so desired forirradiation of an object through the k-space region as described above.This could be, by way of example, an appropriate laser source. Such alaser source could be chosen to be applicable to applications ofMulti-photon Fluorescence Microscopy.

By way of further example, it will be appreciated that referring againto FIG. 18 an excitation application is illustrated at 1800 inaccordance with an aspect of the present invention that can be employedwith the systems and processes previously described. A k-space system isadapted in accordance with the present invention having a light systemthat includes a Laser light source, such as (but not limited to)Ti:Sapphire Mode-Locked Lasers, and/or Nd:YLF Mode-Locked Pulsed Lasers.Such Lasers can be employed to provide for epi-illumination ortrans-illumination as described herein (or other type). The use of sucha Laser (or any other) also enables wave-guide illumination in which theexcitation wavelength is introduced onto a planar surface supporting theobject under test, such that evanescent-wave coupling of the Laser lightcan excite fluorophores within the object in accordance with the desiredparameters of Multi-photon Fluorescence Microscopy. For example, theLaser light can be (but not limited to) provided at about a right angleto a substrate on which the object lies. The Laser source (orcombinations thereof) can emit light for a predetermined time periodand/or be controlled in a strobe-like manner emitting pulses at adesired rate. Automated and/or manual analysis can be performed on theobject during (and/or thereabout) the excitation period. It is notedthat the excitation application described herein can apply to variousprocesses. In one aspect, this includes a process wherein Multi-photonFluorescence Microscopy is the desired result. The present invention maybe employed in any configuration desired (e.g., upright or inverted, orany other disposition such that the fundamental advantage of the opticaldesign is employable.)

Since excitation in multi-photon microscopy occurs at the focal point ofa diffraction-limited microscope it provides the ability to “opticallysection” thick biological specimens in order to obtain three-dimensionalresolution. These “optical sections” are acquired, typically, by rasterscanning the specimen in the x-y plane, and “building” a full“three-dimensional image” which is composed by scanning the specimen atsequential z positions in series. Multi-photon fluorescence is useful,for example, for probing selected regions beneath the specimen surface.This is because the position of the focal point can be accuratelydetermined and controlled.

The lasers commonly employed in optical microscopy are high-intensitymonochromatic light sources, which are useful as tools for a variety oftechniques including optical trapping, lifetime imaging studies, photobleaching recovery, and total internal reflection fluorescence. Inaddition, lasers are also the most common light source for scanningconfocal fluorescence microscopy, and have been utilized, although lessfrequently, in conventional wide field fluorescence investigations.

It will be appreciated that an aspect of the present invention couldincorporate a suitable exciting laser source. It is noted that typicallasers employed for Multi-photon fluorescence currently include theTi:sapphire pulsed laser and Nd:YLF (neodymium: yttrium lithiumfluoride) laser (as well as other available laser sources.) The first isself mode-locked in operation and produces laser light over a broadrange of near-infrared wavelengths with variable pulse widths andgenerally adjustable speed. The exciting laser is joined to the presentinvention through a suitable port. This could be accomplished employingfiber coupling with an optical wave-guide or direct coupling with relaymirrors placed by design to direct the laser energy through the k-spaceregion to the object.

A typical multi-photon fluorescence microscope incorporates a detectorsystem (e.g., a filtered photo multiplier or photodiode or other suchdetector to the laser wavelength) disposed in concert with an x-y rasterscanning device which can rapidly deflect the focused laser beam acrossthe objective field. Digital images collected by the microscope areprocessed and analyzed by a computer and associated software processingto assemble three-dimensional reconstructions from the “opticalsections.” These images display typical of the image sensor-visualmicroscope combination. Modem Multi-photon fluorescence microscopy hasbecome a preferred technique for imaging living cells and tissues withthree-dimensionally resolved fluorescence imaging since two-photonexcitation, which occurs at the focal point of the microscope, minimizesphoto bleaching and photo damage (the ultimate limiting factors inimaging live cells.) This in itself allows investigations on thickliving tissue specimens that would not otherwise be possible withconventional imaging techniques.

The mechanisms that enable sophisticated multi-photon fluorescencemicroscopy result from two-photon and three-photon excitation, forexample. These occur when two or three photons are absorbed byfluorophores in a quantitized event. Photons can, in this way beabsorbed at high enough photon by combining their energies to force anelectronic transition of a fluorosphore to the excited state.

FIGS. 19-20 illustrate beam-scanning technologies that can be applied toincrease excitation yields in accordance with an aspect of the presentinvention. With respect to FIG. 19, an epi-fluorescence system 1900 isillustrated that includes a resolution lens 1910 and matching lens 1920that are adapted to provide diffraction limited mapping of pixels from acamera/sensor 1930 to about the size of diffraction spots associatedwith the resolution lens 1910 as previously described. A fold mirror1940 can be provided to direct energy from the matching lens 1920 to thecamera/sensor 1930. A beam scanner 1950 is provided to increase or focusthe amount of energy from an excitation source 1960 that is received bythe camera/sensor 1930. A beam expander 1970 can also be includedbetween the beam scanner 1950 and the excitation source 1960, wherein abeam splitter 1980 is employed to enable object illumination and todirect energy to the camera/sensor 1930.

In this aspect of the present invention, energy from the excitationsource 1960 is applied in a beam diameter or dimension that is about thesize of a diffraction-limited spot. In this manner, energy from thesource 1960 is intensified at the pixel level and thus greatly increasesthe amount of energy received by the sensor 1930. Since a fullyspatially quantised and resolved system 1900 is being employed, thereare advantages to providing a fluorescent excitation where theexcitation energy substantially matches the optical spatialquantisation. In general, the following considerations can be applied:

1. Maintain current preservation of spatial information.

2. There is substantially no “spillage” of excitation photons ontonearby pixels, thereby greatly reducing the amount of pixel fluorescentcross talk.

3. Rather than exciting the entire field of view, a single pixel (orsubset) is concentrated on, thereby providing a fluorescent yieldenhancement about equal to the number of pixels in the field of view(FOV). For a modest sensor, this can amount to a million-fold increasein yield, for example.

4. For a scanning excitation source, the system 1900 provides theability to selectively scan the FOV and thereby isolate fluorescentstructures of interest.

5. The excitation dosage can be altered by modulating the excitationsource scan frequency, or by modulating the intensity of the excitationsource, for example.

It is noted that the imaging path is generally unaffected, with theexception that the epi-illumination beam splitter 1980 can be a dichroicmirror highly reflective at the excitation wavelength (usually in thenear-UV or deep-blue range). Also, it is desirable that the resolutionlens 1910 (comparable to a conventional objective lens) should betransparent at the excitation wavelengths, if those wavelengths falloutside of the usual visible pass-band, for example.

It is further noted that yield can be increased in other aspects. Forexample, the excitation source 1960 (e.g., LED) can be pulsed via highcurrent and short duration pulses to provide intense periods ofbrightness yet protect the source from excess power dissipation. Thiscan be achieved with custom circuits that generate the pulses or viacommercially available LED driver chips (e.g., Analogic Technologies 500mA charge pump). These chips are high current charge pumps that cangenerate a large current through an LED in a very short time to providea pseudo-flash. Thus, the system 1900 can synchronize the high-powerstrobing of an LED with a scan step frequency of a fluorescencemicroscope or system 1900, and significantly pump up the fluorescenceyield. Typically the chips provide a 500 mA pulse to the LEDs, whereasan LED is typically run at 20 mA continuous, for example.

It is also noted that beam scanning can occur at varying degrees ofoffset, wherein images taken at different scanning offsets can becombined via software to mitigate positional uncertainty for thediffraction-limited spots. In one example, two flying-spot images couldbe captured for a given specimen. The first image would be with theprojected illumination lining up with the projected pixels. The secondimage would be taken with the projected illumination lining up with thespaces between the pixels. These images can be referred to as full- andhalf-step images. The two images would then be digitally processed toform a resultant final image to reduce positional uncertainty.

FIG. 20 illustrates example scanning technologies that can be employedwith the system depicted in FIG. 19. It is to be appreciated that anyscanning system that promotes focusing of energy on a single pixel or asmall subset of pixels is considered to be within the scope of thepresent invention. At 2010, orthogonal mirrors driven via servos,galvanometers or other motors, can be arranged to steer an excitationsource around the epi-illumination path described above with respect toFIG. 19. This type scanning includes possible advantages of low cost andgood optical quality with possible disadvantages of relatively low speedand a folded optical path. At 2020, orthogonal optical wedges can beemployed and arranged to rotate about the optical path, and to cause arefractive shift in the direction of the excitation beam. This typescanning can include possible advantages of an in-line optical path andmoderate speed while possible disadvantages may include refractivedistortions. At 2030, electro-optic modulator crystals can be employedfor scanning. These can include possible advantages of being fast andin-line with the optical path with possible disadvantages that includeexpense, high voltages, and small path divergences.

FIG. 21 illustrates an example system for integrating or retrofittingpixel-mapping components in accordance with an aspect of the presentinvention. In this aspect, portions of a conventional microscopic system2100 include an objective lens and stage at 2110 that is illuminatedfrom a sub-stage lighting module 2120. Light or energy from theobjective 2110 is directed through a telan lens and fold mirror at 2130that is again magnified via an eyepiece 2140. The system 2100 includeswhat is referred to as infinity space 2150 which can enable modules tobe inserted into the space to perform functions such as auto-focus,epi-illumination, fluorescence, phase encoding, filtering and so forth.In this aspect of the present invention, a camera (or sensor) 2160 isassociated with a matching lens 2170 that maps a diffraction limitedspot size of the objective 2110 to about the size of a pixel at thecamera or sensor. A beam splitter 2180 is employed to direct or divertsome energy from the infinity space 2150 to the matching lens 2170 andcamera 2160.

In general, conventional microscope systems are designed so that thereis an “infinity path” or space 2150 between the objective lens 2110 andthe telan 2130/eyepiece 2140 lens assembly. This space 2150 isconventionally used for additions such as epi-illumination, wavelengthfiltering, and so forth. Addition of a digital camera to a conventionalsystem is almost universally achieved by the addition of a lens systemwithin the main optical path, with the lens power arranged so that thecamera sensor “sees” the same field of view as the eyepiece 2140 (andtherefore the observer). It is usual to ensure that the sensor has alarge number of pixels, so that the field of view is finely divided.Unfortunately, this approach ensures that the camera sensor is notoptimally matched to the diffraction-characteristics of the objectivelens 2110, but more to the viewing capabilities of the eyepiece 2140.This leads to a marked drop in potential performance.

The present invention optimizes performance by matching pixelcharacteristics of the camera 2160 to that of the objective 2110 ratherthan to the eyepiece 2140. Thus, the camera 2160, matching lens 2170,and beam splitter 2180 cooperate as an add-on or modifier component to aconventional system's infinity space 2150 (or other space) that enableoptimized digital images to be produced at desired resolutions specifiedby the objective 2110. It is to be appreciated that any sensor and/orlens that is added to (or modifies) a microscopic system (e.g.,industrial, commercial, or medical) to facilitate pixel-scaling ofsensor pixels to about the size of the diffraction-limited spot size isconsidered to be within the scope of the present invention. Also, it isnoted that diffraction performance can be defined by different methodssuch as geometrically via the wavelength of light and the NumericalAperture of the objective, via the Rayleigh criterion, via Airy diskdescription, or via other descriptions such as Sparrow's criterion. Itis to be appreciated that any technique that maps pixel dimensions toabout the size of the diffraction-limited performance of a lens isconsidered to be within the scope of the present invention. Thefollowing describes some example implementations for constructingpixel-mapping components in accordance with the present invention.

In general, light or energy diverted from the infinity path 2150 by thebeam splitter 2180 is directed towards the matching lens 2170, andthereby allowed to fall onto pixels that comprise a sensor or camera2160. The matching lens' power (focal length) should be designed toclosely match the object-plane diffraction-limited spot size to thepixel dimensions, thereby achieving an optimized configuration atdesired resolutions specified by the objective lens 2110. Given thatconventional objective lenses have a range of powers and numericalapertures, it is convenient in one aspect, to select the matching lensto perform for a range of objectives. This would have the effect ofcausing a conventional microscope that is optimized for a range ofmagnifications, concurrently becoming optimized for a range ofresolutions. As can be appreciated, the matching lens 2170 could beprovided in a multiple lens configuration, wherein the lenses in theconfiguration are mechanically switched (in or out) to match thecapabilities of differing objective lenses 2110 (e.g., turret ofmatching lenses adjusted when the objective lens 2110 is changed—turretcould also be synchronized with objective lens turret).

Consider the following sensor examples:

-   -   1. State-of-the-art sensor with 2.7 micron (um) pixels    -   2. Current “good” sensor with 5.0 micron (um) pixels    -   3. Low-cost sensor with 7.5 micron (um) pixels

For three of the most commonly used objective lenses (can also be custommanufactured lenses), the following performance characteristics mayapply: Magnification NA Resolution 10x 0.25  1.0 um 20x 0.40 0.625 um40x 0.65 0.384 um 100x (oil) 1.25 0.200 um

(It is noted that the “magnification” listed in the above chart is validonly for a telan/tube lens of focal length 150 mm).

From the above example lens and sensor characteristics, the informationto create matching lens prescriptions is provided in the followingexamples: Pixel size Resolution Magnification Lens focal length 2.7 um 1.0 um 2.7x 40.5 mm 0.625 um 4.3x 32.4 mm 0.384 um 7.0x 26.4 mm 0.200um 13.5x 20.3 mm 5.0 um  1.0 um 5.0x 75.0 mm 0.625 um 8.0x 60.0 mm 0.384um 13.0x 48.8 mm 0.200 um 25.0x 37.5 mm 7.5 um  1.0 um 7.5x 112.5 mm 0.625 um 12.0x 90.0 mm 0.384 um 19.5x 73.2 mm 0.200 um 37.5x 56.3 mm

The beam splitter 2180 can be any of the following examples:

-   -   1. a beam splitting cube (polarizing or non-polarizing) with        symmetric (e.g., 50:50) or asymmetric (e.g., 70:30) splitting        ratios    -   2. a plane beam splitter, antireflection-coated on one surface,        and coated on the other surface to provide a suitable splitting        ratio (symmetric or asymmetric)    -   3. a thin pellicle.

As can be appreciated, the above sensors and lenses were chosen forillustrative purposes. Thus, any sensor and/or lens combination thatscales or maps diffraction limited performance of a lens to about thesize of a pixel dimension (e.g., pitch) is considered to be within thescope of the present invention.

What has been described above are preferred aspects of the presentinvention. It is, of course, not possible to describe every conceivablecombination of components or methodologies for purposes of describingthe present invention, but one of ordinary skill in the art willrecognize that many further combinations and permutations of the presentinvention are possible. Accordingly, the present invention is intendedto embrace all such alterations, modifications and variations that fallwithin the spirit and scope of the appended claims.

1. A system for generating a digital image, comprising: an opticalsystem having at least one objective lens for microscopic imaging of asample; a sensor having a plurality of pixels to generate an image forthe optical system; and a matching lens associated with the sensor toscale the pixels to about a size of a diffraction-limited parameterassociated with the objective lens.
 2. The system of claim 1, the sensoris associated with a digital camera.
 3. The system of claim 1, theoptical system further comprising an infinity path to enable receivingthe image at the sensor.
 4. The system of claim 3, further comprising abeam splitter to direct the image to the sensor.
 5. The system of claim4, the beam splitter, the matching lens, and the sensor are adapted tothe infinity path to enable retrofitting of a digital imager into anexisting system.
 6. The system of claim 3, the infinity path enables atleast one optical module to be associated with the path.
 7. The systemof claim 6, the optical module includes at least one of an auto focusmodule, an epi illumination module, a fluorescence module, a phaseencoding module, and a filter module.
 8. The system of claim 1, theoptical system is at least one of an industrial optical system, acommercial optical system, and a medical optical system.
 9. The systemof claim 1, the diffraction-limited parameter is associated with atleast one of a geometrical criterion defined by an energy wavelength anda Numerical Aperture, a Rayleigh criterion, an Airy disk criterion, anda Sparrow's criterion.
 10. The system of claim 1, the matching lens'having a focal length designed to approximate an object-planediffraction-limited spot size with a pixel dimension.
 11. The system ofclaim 1, the matching lens is designed to accommodate a range of powersassociated with a set of objective lenses.
 12. The system of claim 1,further comprising a set of matching lenses that are correlated toprovide diffraction-limited mapping of pixels with a set of objectivelenses.
 13. The system of claim 12, the matching lenses are synchronizedwith the set of objective lenses such that if a different objective lensis selected having a different resolution, a matching lens isautomatically selected to provide diffraction-limited pixel matching.14. The system of claim 1, the pixels have pitch size of about 2 micronsto about 10 microns.
 15. The system of claim 14, the pixels areassociated with a resolution lens having a numerical aperture from about0.1 to about 1.3.
 16. The system of claim 14, the pixels are associatedwith a magnification lens having a magnification from about 2 times toabout 14 times with an associated focal length from about 40 millimetersto about 20 millimeters, the pixels are sized from about 2 microns and 3microns per pixel.
 17. The system of claim 14, the pixels are associatedwith a magnification lens having a magnification from about 5 times toabout 25 times with an associated focal length from about 75 millimetersto about 38 millimeters, the pixels are sized from about 4 microns andabout 6 microns per pixel.
 18. The system of claim 14, the pixels areassociated with a magnification lens having a magnification from about 7times to about 38 times with an associated focal length from about 112millimeters to about 56 millimeters, the pixels are sized from about 7microns and about 8 microns per pixel.
 19. The system of claim 4, thebeam splitter includes at least one of a beam splitting cube, a planebeam splitter, and a thin pellicle.
 20. A method for generating adigital image, comprising: selecting an optical configuration having atleast one objective lens for generating a microscopic image of aspecimen; and adapting a sensor having a plurality of pixels to amatching lens, the sensor and the matching lens adapted to the opticalconfiguration, the matching lens scales the pixels to about a size of adiffraction-limited parameter associated with the objective lens.
 21. Asystem for generating a digital image, comprising: means for generatinga microscopic image of a specimen; means for digitally sensing themicroscopic image in an infinity space; and means for matching pixels toabout a size of a diffraction-limited parameter associated with anobjective lens that resolves the microscopic image.